Von Willebrand factor (VWF) binds exposed collagen at sites of blood vessel injury. Type 4 collagen is one of the vascular collagens, found in the basement membrane of endothelial cells. Patients with defects in VWF that reduce or eliminate collagen binding experience bleeding, as do patients with genetic defects in collagen 4. Little is known, however, about the mechanism of collagen 4-VWF interactions. The long term goal of this project is to improve understanding of the mechanism of coagulation as initiated by VWF and collagen, and to develop interventions to improve hemostasis in patients with defects in VWF-collagen interactions. The overall hypothesis of this proposal is that the VWF A1 domain interaction with collagen 4 is crucial for normal hemostasis. Specific aim 1 will define the molecular interactions required for VWF binding to collagen 4. In vitro studies will include assessment of the binding site for collagen 4 on VWF using various VWF A1 domain mutations. VWF-collagen 4 interactions will be assessed in the context of platelets under both static and shear conditions. In vivo dissection of this interaction will be performed using murine models to quantify bleeding and thrombosis in the setting of abnormal VWF-collagen 4 interactions. Interactions with collagen 4 will be compared to those seen with collagen 6, another vascular collagen that also binds VWF via the VWF A1 domain. Specific aim 2 will define the molecular interactions required for collagen 4 binding to VWF. VWF binding to fragments containing specific collagen 4 domains will be tested to isolate the region of collagen 4 responsible for VWF interactions. Pathogenic sequence variations in collagen 4 will be examined via whole exome sequencing in samples from a well-characterized population with significant yet unexplained bleeding symptoms. Variants in other vascular collagens will also be examined. This project will use the resources available at the Medical College of Wisconsin and the Blood Research Institute of the BloodCenter of Wisconsin, including access to unique blood samples, expertise in murine hemostasis research, and strong genetics support, to generate key data on the molecular interactions between VWF and collagen 4 in hemostasis. The results of this project will further understanding of the mechanism(s) underlying VWF interactions with exposed collagen 4 and the impact of defects in this interaction. Clarification of the molecular interactions between VWF and collagen 4 will ultimately lead to a better understanding of the biology of hemostatic defects and facilitate translation of these findings to improved treatment for affected patients.